Although carbohydrates are just as important constituents of living bodies as nucleic acids and proteins, their structures and mechanisms are not as well understood as those of nucleic acids and proteins. Carbohydrates frequently form polymers having sugar chain in sequence, and bind with proteins or lipids to form extremely complicated composite molecules, glycoconjugates, which are called glycoproteins, glycolipids, and proteoglycans. Nucleic acids and proteins are polymers wherein constituent units of nucleotides or amino acids are linked to one other linearly. In contrast, carbohydrates have a plurality of intramolecular branch points and their constitutional units, i.e., monosaccharides, are linked to each other in various manners, and therefore carbohydrates have complicated structures incomparable to those of nucleic acids or proteins. This structural complexity is one of the major causes of the delay in the study of carbohydrates.
In recent years, since it has been gradually revealed that carbohydrates have a roll in cell recognition, immunity, differentiation, fertilization, aging, canceration, etc., carbohydrates become a target of study that attracts significant attention. Under such circumstances, many attempts have been made to synthesize a sugar chain having natural structure and a novel sugar chain. Automatic synthesis techniques for nucleic acids and proteins have been already established, and these techniques have obviously accelerated the progress of the researches in these fields. Therefore, the establishment of automatic synthesis techniques for sugar chains have been eagerly desired.
Several attempt for automatic synthesis for sugar chains have so far been reported, and their approaches can be roughly classified into two groups. One employs chemical synthesis, which has many problems such as the fact that stereoselective glycosylation reaction has not been well established, and the process is tedious and complicate because of protection and deprotection. Another employs enzymatic synthesis, which requires no protection, and glycosylation reaction can be carried out stereoselectively. Therefore, compared to chemical synthesis, enzymatic synthesis has many advantages. Several methods have been proposed in recent years. It is due to cloning the genes of various glycosyltransferases and economical production of some recombinant glycosyltransferases. In automatic synthesis, a certain carrier (sometimes referred to as a primer) bound sugar residues, which serve as initiators, through linkers that can be cleaved under specific conditions is used as a starting material. The types of glycoconjugate produced depend on the kind of linkers, and they are released from the carrier as oligosaccharides, glucosides, glycopeptides, glycolipids, etc.
As one of the examples of automatic synthesis method of a sugar chain using glycosyltransferases, U. Zehavi et al. have reported on a solid-phase synthesis method using a polyacrylamide gel bound with an aminoethyl group or an aminohexyl group as a solid-phase carrier (see, for example, Carbohydr. Res., 124(1983), 23; and Carbohydr. Res., 228(1992), 255). This method comprises the steps of converting a suitable monosaccharide to 4-carboxy-2-nitrobenzyl glycoside, condensing this glycoside with amino group of the above carrier, elongating the sugar chain by glycosyltransferase using the condensate as a primer, and releasing the elongated sugar chain as oligosaccharide by photolysis. According to this method, however, sugar transfer yield is low, i.e., less than 10%. It has been a common understanding that glycosyltransferase dose not react well with monosaccharide or oligosaccharide bound to a solid-phase carrier and efficient elongation of sugar chain reaction is difficult to achieve. However, U. Zehavi et al. have documented in a recent report that the sugar transfer yield could be improved up to 51% by using a linker between 4-carboxy-2-nitrobenzyl glycoside and the solid-phase carrier having a long chain such as hexamethylene, and octamethylene, etc. (see, for example, React. Polym., 22(1994), 171; Carbohydr. Res., 265(1994), 161). However, even this method cannot achieve satisfactory yields.
As another example, C.-H. Wong et al. have reported a method wherein a sugar chain is elongated using glycosyltransferases and aminated silica bound a group represented by the following formula
(wherein Ac is an acetyl group and Boc is a t-butoxycarbonyl group) as a primer, and the elongated sugar chain was released in the form of a glycopeptide by hydrolysis of α-chymotrypsin (see, for example, J. Am. Chem. Soc., 116(1994), 1136). However, the yield of sugar-chain elongation reaction using glycosyltransferase is unsatisfactory at 55 to 65%.
Furthermore, C.-H. Wong et al. revised the group to be bound to the solid phase as aminated silica to represented by the following formula
(wherein Ac is an acetyl group) and reported a method wherein the sugar chain was elongated using glycosyltransferases and released by hydrazinolysis. They also reported that the enzymatic glycosylation reaction was proceeded almost quantitatively (see, for example, J. Am. Chem. Soc., 116(1994), 11315). In this method, the elongated sugar chain is released in the form of a 6-carbohydrazide hexanol glucoside.
M. Meldal et al. have reported a method wherein a sugar chain was elongated using glycosyltransferases and a polymer gel of mono- and diacryloyl compound of diaminated poly(ethylene glycol) having a group represented by the following formula
(wherein Ac is an acetyl group) as a primer and the elongated sugar chain was released in the form of a glycopeptide using trifluoroacetic acid. According to their report, the transglycosylation reaction is proceeded almost quantitatively (see, for example, J. Chem. Soc., Chem. Commun., 1849 (1994)). The peptide sequence in the glycopeptide obtained by this method is Asn (asparagine)-Gly (glycine), and the glycine residue at the C-end is a glycinamide residue, and therefore it differs from typical glycopeptides. C.-H. Wong et al. have also reported a method for releasing typical glycopeptides synthesized on a solid-phase carrier. In this method, aminated silica is used as a solid-phase carrier introduced a group represented by the following formula as a primer
(wherein Fmoc is (9-fluorenylmethyl)oxycarbonyl)), the peptide chain is elongated using Fmoc-amino acids and Fmoc-Thr(βGlcNAc)-OH, after peptide elongation protecting groups on the peptide chain are eliminated, the sugar chain is elongated by glycosyltransferase to the above-mentioned N-acetylglucosamine residue, and resultant glycopeptide is released by tetrakis(triphenylphosphine)palladium treatment (see, for example, J. Am. Chem. Soc., 119(1997), 8766). The yield of the obtained glycopeptide estimated from the amino acid initially introduced to the solid-phase carrier is less that 10%, which is unsatisfactory.
T. Norberg et al. have reported a method wherein a sugar chain was elongated using Sepharose 6B (manufactured by Amersham Pharmacia Biotech) bound a group represented by the following formula as a primer and glycosyltransferase,
and the elongated sugar chain is released by treatment with bromine or ammonia/ammonium borate (see, for example, Carbohydr. Res., 319(1999), 80). In this method, the enzymatic transglycosylation reaction proceeds quantitatively, so there is no problem with the yield. However, this method is uneconomical because expensive 3,4-diethoxy-3-cyclobutene-1,2-dione is used for producing the primer. The above-described methods have drawbacks such that the transglycosylation reaction yield is unsatisfactory, and/or an immobilized glycosyltransferase cannot be applied since the sugar-chain elongation reaction is carried out on a water-insoluble carrier. In sugar chain elongation by glycosyltransferases, the use of immobilized glycosyltransferases that permit repetitive use are desirable, since glycosyltransferases are still very expensive, though mass production of glycosyltransferases by genetic recombination techniques are becoming available. In order to use an immobilized glycosyltransferase, the sugar-chain elongation reaction should be proceeded not on a water-insoluble carrier but on a water-soluble carrier.
S. Roth et al. have disclosed a method as follows (see, for example, Japanese Unexamined Patent Publication No. 1993-500905). Saccharide, sugar acceptor for a glycosyltransferase, is bound to a solid-phase carrier to form an affinity adsorbent, and a glycosyltransferase is then adsorbed to the above adsorbent by contacting with a tissue extract containing a glycosyltransferase that can recognize the sugar acceptor. Thereafter, the glycosyltransferase transfers sugar residue from sugar nucleotide to the acceptor on the absorbent and elutes from the adsorbent by contacting with a solution containing a sugar nucleotide which the glycosyltransferase can use as a sugar donor. Furthermore, by repetition of contacting the resultant one sugar residue elongated sugar chain on the absorbent with a tissue extract containing another glycosyltransferase that can recognize the elongated sugar acceptor and the similar elution procedure, a desired sugar chain can be synthesized on a solid-phase carrier. However, no concrete data demonstrating the effectiveness of this method is provided. Furthermore, no methods for releasing the elongated sugar chain from the solid-phase carrier are disclosed.
C.-H. Wong et al. have also reported a method for elongating a sugar chain on a water-soluble carrier wherein a water-soluble polymer, acrylamide/acrylic acid/N-isopropylacrylamide copolymer bound a group represented by the following formula to acrylamide residue in this copolymer
(wherein Ac is an acetyl group) is used as a primer, the sugar chain is elongated by glycosyltransferases and released by the treatment with cerium (IV) diammonium nitrate (see, for example, Adv. Synth. Catal., 343(2001), 675). The proportion of acrylic acid in the copolymer primer used in this method is 4%, and therefore this primer differs from that of the present invention. In this method, the enzymatic transglycosylation reaction progresses at 80 to 90% yield, and the elongated sugar chain is released in the form of a p-formylphenol glucoside. However, this method has drawbacks such that column chromatography using organic solvents is required to purify the released p-formylphenol glucoside, and in some cases obtained p-formylphenol glucoside is not so stable.
The present inventors have reported a method for elongating a sugar chain on a water-soluble carrier wherein polyacrylamide bound a group represented by the following formula
(wherein Ac is an acetyl group) to every fifth amide nitrogen atom in it is used as a primer, the sugar chain is elongated by glycosyltransferases, and the elongated sugar chain is released in the form of an oligosaccharide by hydrogenolysis (see, for example, Tetrahedron Lett., 35(1994), 5657; Carbohydr. Res., 305(1998), 443).
The present inventors have also reported a method for elongating a sugar chain on a water-soluble carrier wherein polyacrylamide bound a group represented by the following formula
(wherein Ac is an acetyl group) to an amide nitrogen atom of amide moiety is used as a primer, the sugar chain is elongated glycosyltransferases, and the elongated sugar chain is released in the form of a 6-aminohexanol glucoside by hydrolysis of α-chymotrypsin (see, for example, Tetrahedron Lett., 36(1995), 9493; Carbohydr. Res., 305(1998), 443).
According to these reports by the present inventors, glycoconjugates can be efficiently synthesized by free enzymes; however, as described later, when an immobilized enzyme is used, the production efficiency is unsatisfactory. The present inventors have also reported a method for elongating a sugar chain on a water-soluble carrier wherein polyacrylamide bound peptide residue linked monosaccharide residue to functional group on side chain of amino acid in this peptide residue through a linker which has a desired length and comprises an amino acid residue or peptide residue having a cleavage site for a certain protease for example a group represented by the following formula
(wherein Ac is an acetyl group) is used as a primer, a certain protease dose not have cleavable site in the above-mentioned peptide which bound directly polyacrylamide, the sugar chain elongation is initiated on the above-mentioned monosaccharide by glycosyltransferases, and the elongated sugar chain is released in the form of a glycopeptide using a appropriate protease hydrolysis (see, for example, Japanese Unexamined Patent Publication No. 2001-220399).
The present inventors have also reported a method which uses a primer comprising a residue represented by the following formula (VIII)
(wherein R13 and R14 are independently H, a monosaccharide residue or an oligosaccharide residue; R15 is a C6-20 alkyl group or C6-20 alkenyl group; and R16 is a C5-19 alkylene group) bound to a side chain of a water-soluble polymer. In this method, the sugar chain is elongated by glycosyltransferases, an resultant oligosaccharide residue is transferred in the presence of a ceramid from the polymer having elongated sugar chain to the ceramide by ceramide glycanase, and then liberated as a sphingoglycolipid (see, for example, Japanese Unexamined Patent Publication No. 1998-251287).
In the above-described methods, polyacrylamide is disclosed as one example of water-soluble polymer but no examples are disclosed when acrylic acid is used. Furthermore, when a polyacrylamide is used as a water-soluble polymer, if the transglycosylation reaction is proceeded by immobilized glycosyltransferases, its efficiency is unsatisfactory as described later. If the latter primer is used, when gel filtration chromatography and ultrafiltration are performed to remove by-product nucleotides and unreacted sugar nucleotides after the transglycosylation reaction, recovery of the primer is not always satisfactorily high.
One of the main objects of the present invention is to provide a compound that is suitable and useful as a primer for automatic synthesis of various kinds of glicoconjugates, and a method for producing glycoconjugate using the compound.